Nitrate (NO3) profiles in semiarid unsaturated zones archive land use change (LUC) impacts on nitrogen (N) cycling with implications for agricultural N management and groundwater quality. This study quantified LUC impacts on NO3 inventories and fluxes by measuring NO3 profiles beneath natural and rainfed (nonirrigated) agricultural ecosystems in the southern High Plains (SHP). Inventories of NO3-N under natural ecosystems in the SHP normalized by profile depth are extremely low (2-10 kg NO3-N/ha/m), in contrast to those in many semiarid regions in the southwestern U.S. Many profiles beneath cropland (9 of 19 profiles) have inventories at depth that range from 28-580 kg NO3--N/ha/m (median 135 kg/ha/m) that correspond to initial cultivation, dated using soil water Cl. These inventories represent 74% (median) of the total inventories in these profiles. This NO3 most likely originated from cultivation causing mineralization and nitrification of soil organic nitrogen (SON) in old soil water (precultivation) and is attributed to enhanced microbial activity caused by increased soil wetness beneath cropland (median matric potential -42 m) relative to that beneath natural ecosystems (median -211 m). The SON source is supported by isotopes of NO3 (delta15N: +5.3 to +11.6; delta18O: +3.6 to +12.1). Limited data in South Australia suggest similar processes beneath cropland. Mobilization of the total inventories in these profiles caused by increased drainage/ recharge related to cultivation in the SHP could increase current NO3-N levels in the underlying Ogallala aquifer by an additional 2-26 mg/L (median 17 mg/L).